Mutation screening using formalin-fixed paraffin-embedded tissues: a stratified approach according to DNA quality

Abstract

DNA samples from formalin-fixed paraffin-embedded tissues are highly degraded with variable quality, and this imposes a big challenge for targeted sequencing due to false positives, largely caused by PCR errors and cytosine deamination. To eliminate false positives, a common practice is to validate the detected variants by Sanger sequencing or perform targeted sequencing in duplicate. Technically, PCR errors could be removed by molecular barcoding of template DNA prior to amplification as in the HaloPlexHS design. Nonetheless, it is uncertain to what extent variants detected using this approach should be further validated. Here, we addressed this question by correlating variant reproducibility with DNA quality using HaloPlexHS target enrichment and Illumina HiSeq4000, together with an in-house validated variant calling algorithm. The overall sequencing coverage, as shown by analyses of 70 genes in 266 cases of large B-cell lymphoma, was excellent (98%) in DNA samples amenable for PCR of ≥400 bp, but suboptimal (92%) and poor (80%) in those amenable for PCR of 300 bp and 200 bp respectively. By mutation analysis in duplicate in 93 cases, we demonstrated that 20 alternative allele depth (AAD) was an optimal cut-off value for separating reproducible from non-reproducible variants in DNA samples amenable for PCR of ≥300 bp, with 97% sensitivity and 100% specificity. By cross validation with a previously established targeted sequencing protocol by Fluidigm-PCR and Illumina MiSeq, the HaloPlexHS protocol was shown to be highly sensitive and specific in mutation screening. To conclude, we proposed a stratified approach for mutation screening by HaloplexHS and Illumina HiSeq4000 according to DNA quality. DNA samples with good quality (≥400 bp) are amenable for mutation analysis with a single replicate, with only variants at 15-20 AAD requiring for further validation, while those with suboptimal quality (300 bp) are better analysed in duplicate with reproducible variants at >15 AAD regarded as true genetic changes.

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“…This was essentially carried out as described previously [31]. Briefly, 100 ng genomic DNA was subjected to targeted enrichment of 70 genes (Table S1), which are recurrently mutated in aggressive B-cell lymphomas using a customised HaloPlexHS probe library (Agilent Technologies).…”

“…The pooled libraries were sequenced on an Illumina HiSeq4000 (2 × 150 bp end sequencing protocol) or HiSeq2500 (Rapid Run Mode 2 × 150 bp end sequencing protocol). As stipulated by our previous study, DNA samples amenable for PCR of ≥400 bp genomic fragments were investigated in a single replicate, while those amenable for PCR of 300 bp were analysed in duplicates, with reproducible variants being considered as a true change [31].…”

“…Sequence data analysis and variants calling were performed using a previously validated in-house protocol [31]. Briefly, SNV were called using UnifiedGenotyper with no downsampling [32].…”

“…The resulting variants were further scrutinised by reviewing the bam file to eliminate potential PCR and sequence artefacts. Only variants above the cut-off value (20 alternative allele depth for DNA samples amenable for PCR of ≥400 bp, 15 alternative allele depth in both replicates for DNA samples amenable for PCR of 300 bp) were considered to be a true change [31]. Finally, extensive search of COSMIC database and published literature was carried out to retain those known and confirmed to be somatic variants.…”

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“…A panel of 70 genes that are recurrently mutated in aggressive B-cell lymphomas were investigated for mutation by targeted sequencing using HaloPlexHS target enrichment (Agilent Technologies, Santa Clara, CA, USA) and Illumina HiSeq sequencing, as described previously. 11 This process was carried out for participants who had DNA available of adequate quantity and quality. Duplicate experiments were done for samples of lower quality, including all those with quality control PCR showing amplification of 300 bp or fewer genomic fragments, and only those mutations that were repro ducible in both experiments were reported.…”